Abstract The goal of this SBIR project is to develop an innovative SA-PCPE-PCR technology that can simultaneously genotype multiple mutated mononucleotide STRs in a vast background of wild-type DNA, which will be employed to develop an assay for liquid-biopsy of high-frequency microsatellite instability (MSI-H) cancer. The hallmark of MSI-H is extensive instability in short tandem repeat (STR), which alters the repeat number. Examination of five mononucleotide STRs is currently recommended for the MSI classification. Targeted cancer treatment is emerging as an effective approach to cancer care because it allows doctors to select the right treatment for the right patient at the right time based on a genetic understanding of tumor. Because MSI-H has different profiles than microsatellite stable (MSS) tumors, their treatment differs from each other. Recently, FDA approved the first two drugs that are tailored to MSI-H treatment. For example, FDA granted an accelerated approval of Keytruda to a treatment for patients with MSI-H. Until then, FDA approved cancer treatments based on where in the body the cancer started?for example, lung or breast cancers. Keytruda is the first approved drug based on a tumor's biomarker (MSI-H) without regard to the tumor's original location. Liquid biopsy is a method enabling doctors to discover a range of information about a tumor through a blood sample, and detecting mutations in cell-free DNA (cfDNA) is emerging as the method of choice of liquid biopsy. Targeted treatment is one of its most important application areas. Because of its values to targeted cancer treatment, various liquid biopsy assays as the accompany test for targeted treatment have been or are being developed. For example, FDA has approved liquid biopsy tests for targeted EGFR treatment. Clearly, liquid biopsy will be equally valuable to targeted MSI-H treatment. However, no liquid biopsy test is available for targeted MSI-H cancer treatment because of the lack of technologies that can detect the biomarker of MSI-H in a sensitive, specific, and cost-effective manner. Herein, we propose a novel method termed SA-PCPE-PCR to address this unmet need. This method solves two of the fundamental problems plaguing the detection of mutated STRs in cfDNA. First, it enriches multiple mutated STRs simultaneously prior to PCR. This solves the problem of PCR slippages, rendering it possible to genotype mutated STRs in a vast background of wild-type DNA using fragment analysis. Second, it manipulates fragment size of amplicons by introducing size-tags, creating a sufficient difference in length of the amplicons. This solves the size constraint problems imposed by cfDNA, making it possible to genotype multiple STRs based on the unique size of each amplicon. A SA-PCPE-PCR assay consists of three steps. First, a DNA sample is subjected to SA-PCPE, which preferentially produces amplifiable (long) extension products from mutant alleles (enriching mutants prior to PCR), while introducing size-tags to these extension products. Second, multiplexed PCR is performed with extension products as templates, but only long extension products are amplified. Finally, size-adjusted amplicons are fragment analyzed by a conventional DNA sequencer to identify mutated STRs. In this study, we will first develop a SA-PCPE-PCR assay to detect five mutated mononucleotide STRs simultaneously in a large background of wild-type DNA. Then, we will assess the detection limit and reproducibility of the assay. Finally, we will assess the clinical detection sensitivity and specificity of the assay. Clearly, success of this project can provide a liquid biopsy assay that may transform the landscape of personalized MSI-H cancer medicine.